Methods for the diagnosis of bacterial vaginosis

ABSTRACT

The present invention relates to methods for the diagnosis of bacterial vaginosis based on an analysis of a patient sample. For example, patient test samples are analyzed for the presence or absence of one or more  lactobacilli  and two or more pathogenic organisms. The presence or absence of one or more  lactobacilli  and two or more pathogenic organisms may be detected using PCR analysis of nucleic acid segments corresponding to each target organism. The quantity of the target organisms can then be used to determine a score which is indicative of a diagnosis of bacterial vaginosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application of PCT/US2010/056983,filed Nov. 17, 2010, which claims priority from U.S. ProvisionalApplication No. 61/266,338, filed Dec. 3, 2009.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 21, 2012, isnamed sequence.txt and is 6 KB.

FIELD OF THE INVENTION

The present technology relates generally to the field of medicaldiagnostics. In particular, the present technology relates to methods ofdetecting the presence or absence of bacteria associated with bacterialvaginosis, and determining a diagnostic score based on the presence orabsence of the bacteria.

BACKGROUND OF THE INVENTION

The following description is provided to assist the understanding of thereader. None of the information provided or references cited is admittedto be prior art to the present invention.

Vaginitis is the most common gynecological problem in adult women.Infectious vaginitis presents itself in three primary forms: bacterialvaginosis, candidal vaginitis, and trichomonas vaginitis. Bacterialvaginosis, which affects up to 25% of American women in normal clinicalpopulations, is nearly twice as common as candida and is the most commonform of vaginal infection. Bacterial vaginosis is caused by areplacement of the normal vaginal flora with facultative anaerobicbacteria. Typically, the symptoms of bacterial vaginosis arenon-specific and differential diagnosis is problematic.

Complications associated with bacterial vaginosis represent a majorhealth care cost burden. For example, obstetric complications ofbacterial vaginosis include preterm labor/birth, low birth weightbabies; premature rupture of the amniotic membranes; amniotic fluidinfections; postpartum endometritis; and chorioamnionitis. Also,bacterial vaginosis is suspected of being one of the many causes ofcerebral palsy. In addition, gynecologic complications of bacterialvaginosis include postoperative infections; pelvic inflammatory disease(PID); abnormal cervical cytology, increased susceptibility to sexuallytransmitted diseases (STDs), and post-hysterectomy infections.Furthermore, bacterial vaginosis may potentially be a cofactor withhuman papilloma virus in the development of cervical intraepithelialneoplasia (CIN), a precursor of cervical cancer.

Diagnosis of BV has traditionally been performed using the Amsel'scriteria, which include any three of: abnormal vaginal discharge, pH ofmore than 4.5, foul odor after the addition of potassium hydroxide, orpresence of clue cells in Gram stain; or by the calculation of a Nugentscore. The Nugent score is determined from a microscopic test measuringthe relative number of Lactobacillus ssp., Gardnerella vaginalis,Bacteroides ssp., and Mobiluncus-like species.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for diagnosingbacterial vaginosis in a subject, the method comprising: (a) determininga single diagnostic score using the levels of one or more lactobacilliand two or more pathogenic organisms in a sample from the subject; and(b) comparing the diagnostic score for the individual to a referencescore to determine the presence of bacterial vaginosis, wherein saidsingle diagnostic score is determined by finding the ratio of alogarithmic function of the levels of the one or more lactobacilli and alogarithmic function of the levels of the two or more pathogenicorganisms. In one embodiment, the sample is a vaginal swab.

In one embodiment, the logarithmic function applied to the one or morelactobacilli comprises determining the sum of the logarithm of the levelof each lactobacilli used, and the logarithmic function applied to thetwo or more pathogenic organisms comprises determining the sum of thelogarithm of the level of each pathogenic organism used. In oneembodiment, the reference score is about 0.2, and a diagnostic scoreless than about 0.2 is indicative of the presence of bacterialvaginosis.

In one embodiment, the logarithmic function applied to the one or morelactobacilli comprises determining the logarithm of the sum the levelsof each lactobacilli used, and the logarithmic function applied to thetwo or more pathogenic organisms comprises determining the logarithm ofthe sum of the levels of each pathogenic organism used. In oneembodiment, the reference score is about 0.2, and a diagnostic scoreless than about 0.2 is indicative of the presence of bacterialvaginosis.

In one embodiment, the levels of one or more lactobacilli and two ormore pathogenic organisms in the sample are determined by detectingnucleic acids indicative of the one or more lactobacilli and two or morepathogenic organisms. In one embodiment, the detecting is by PCR,RT-PCR, or nucleic acid hybridization. In one embodiment, the detectingcomprises amplifying a fragment from each of the one or morelactobacilli and two or more pathogenic organisms in the sample, ifpresent. In one embodiment, the fragment is a fragment of a 16Sribosomal RNA gene. In an illustrative embodiment, the detecting isaccomplished using the TaqMan® PCR detection system.

In one embodiment, the one or more lactobacilli are selected from thegroup consisting of Lactobacillus acidophilus, Lactobacillus crispatus,Lactobacillus jensenii, Lactobacillus iners and Lactobacillus vaginalis.In one embodiment, the levels of one or more lactobacilli are detectedusing primers capable of detecting Lactobacillus spp. In one embodiment,at least one of the primers capable of detecting Lactobacillus spp. areselected from the group consisting of SEQ ID NOs: 8-9 or complementsthereof.

In one embodiment, the levels of one or more lactobacilli are detectedusing one or more primer pairs capable of detecting Lactobacillusacidophilus, Lactobacillus crispatus, and Lactobacillus jensenii. In oneembodiment, at least one of the primers capable of detectingLactobacillus acidophilus and Lactobacillus crispatus are selected fromthe group consisting of SEQ ID NOs: 1-2 or complements thereof. In oneembodiment, at least one of the primers capable of detectingLactobacillus jensenii are selected from the group consisting of SEQ IDNOs: 4-5 or complements thereof. In one embodiment, at least one of theprimers capable of detecting Lactobacillus vaginalis are selected fromthe group consisting of SEQ ID NO: 20-21 or complements thereof.

In one embodiment, at least one of the two or more pathogenic organismsis selected from the group consisting of Atopobium vaginae, Megasphaerassp., and Gardnerella vaginalis. In one embodiment, at least one of theprimers capable of detecting Atopobium vaginae are selected from thegroup consisting of SEQ ID NOs: 11-12 or complements thereof. In oneembodiment, at least one of the primers capable of detecting Megasphaerassp. are selected from the group consisting of SEQ ID NOs: 14-15 orcomplements thereof. In one embodiment, at least one of the primerscapable of detecting Gardnerella vaginalis are selected from the groupconsisting of SEQ ID NOs: 17-18 or complements thereof.

In specific embodiments of any of the foregoing the logarithmic functionmay be any one of Algorithms 1-10 identified herein. The specificorganisms identified in the algorithms are intended merely as examples.The measured level of any of the lactobacilli species may be substitutedfor the measured level of any other non-pathogenic lactobacillus. And,the measured level of any of the pathogenic bacteria may be substitutedwith the level of any other pathogenic bacteria.

In one aspect, the present invention provides a kit for diagnosingbacterial vaginosis comprising a primer pair for amplifying a fragmentof a nucleic acid from one or more lactobacilli and primer pairs foramplifying fragments of nucleic acids from two or more pathogenicorganisms. In one embodiment, at least one primer pair is selected fromthe group consisting of: SEQ ID NOs: 1/2, SEQ ID NOs: 4/5, SEQ ID NOs:8/9; SEQ ID NOs: 11/12; SEQ ID NOs: 14-15; and SEQ ID NOs: 17/18 orcomplements thereof.

In one aspect, the present invention provides a substantially purifiedoligonucleotide having a sequence selected from the group consisting ofSEQ ID NOs: 1-19 or complements thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart showing the percent of swab specimens containingvarious bacterial agents arranged by Nugent Score. The bacterial agentswere detected according to an illustrative embodiment of the invention.

FIG. 2 is a chart showing the mean quantities of bacterial agents inswab specimens from patients arranged by Nugent Score. The bacterialagents were detected according to an illustrative embodiment of theinvention.

DETAILED DESCRIPTION

The present invention provides methods of diagnosing bacterial vaginosis(BV) by detecting in a test nucleic acid sample from the individual oneor more nucleic acid segments corresponding to various bacterial speciesthat are relevant to a diagnosis of BV. In particular embodiments,nucleic acid segments corresponding to lactobacilli, and one or morepathogenic bacteria are detected. This assay can be performed in one ormore subassays to detect the bacterial targets of interest. For example,one subassay detects peroxide-producing lactobacilli (“Assay A”); onesubassay detects all lactobacilli (“Assay B”); one subassay detectspathogenic bacteria Megasphaera spp. and Atopobium vaginae (“Assay C”);and one subassay detects the pathogenic bacteria Gardnerella vaginalis(“Assay D”). This information may be used to determine whether anindividual is suffering from BV. In some embodiments, a diagnostic scorecorresponding to a diagnosis of BV is determined. For example, the scoremay be determined by finding the ratio of a logarithmic function of thelevels of the one or more lactobacilli and a logarithmic function of thelevels of the two or more pathogenic organisms.

In practicing the methods described herein, many conventional techniquesin molecular biology, protein biochemistry, cell biology, immunology,microbiology and recombinant DNA are used. These techniques arewell-known and are explained in, e.g., Current Protocols in MolecularBiology, Vols. I-III, Ausubel, Ed. (1997); Sambrook et al., MolecularCloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989); DNA Cloning: A PracticalApproach, Vols. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis,Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds.(1985); Transcription and Translation, Hames & Higgins, Eds. (1984);Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes(IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; theseries, Meth. Enzymol., (Academic Press, Inc., 1984); Gene TransferVectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring HarborLaboratory, NY, 1987); and Meth. Enzymol., Vols. 154 and 155, Wu &Grossman, and Wu, Eds., respectively.

The definitions of certain terms as used in this specification areprovided below. Unless defined otherwise, all technical and scientificterms used herein generally have the same meaning as commonly understoodby one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a nucleic acid”includes a combination of two or more nucleic acids, and the like.

The term “amplification” or “amplify” as used herein means one or moremethods known in the art for copying a target nucleic acid, therebyincreasing the number of copies of a selected nucleic acid sequence.Amplification may be exponential or linear. A target nucleic acid may beeither DNA or RNA. The sequences amplified in this manner form an“amplicon.” While the exemplary methods described hereinafter relate toamplification using the polymerase chain reaction (“PCR”), numerousother methods are known in the art for amplification of nucleic acids(e.g., isothermal methods, rolling circle methods, etc.). The skilledartisan will understand that these other methods may be used either inplace of, or together with, PCR methods. See, e.g., Saiki,“Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds.,Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al.,Nucleic Acids Res., 2001, 29(11):E54-E54; Hafner et al., Biotechniques2001, 30(4):852-6, 858, 860; Zhong et al., Biotechniques, 2001,30(4):852-6, 858, 860.

The term “complement” as used herein means the complementary sequence toa nucleic acid according to standard Watson/Crick base pairing rules. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence or its complement sequence, and can also be a cDNA. Theterm “substantially complementary” as used herein means that twosequences hybridize under stringent hybridization conditions. Theskilled artisan will understand that substantially complementarysequences need not hybridize along their entire length. In particular,substantially complementary sequences comprise a contiguous sequence ofbases that do not hybridize to a target or marker sequence, positioned3′ or 5′ to a contiguous sequence of bases that hybridize understringent hybridization conditions to a target or marker sequence.

As used herein the terms “diagnose” or “diagnosis” or “diagnosing” referto distinguishing or identifying a disease, syndrome or condition ordistinguishing or identifying a person having a particular disease,syndrome or condition. In illustrative embodiments of the invention,assays and algorithms are used to diagnose bacterial vaginosis in asubject based on an analysis of a sample.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically conducted with probe-length nucleic acid molecules. Nucleicacid hybridization techniques are well known in the art. See, e.g.,Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Press, Plainview, N.Y. Those skilled in theart understand how to estimate and adjust the stringency ofhybridization conditions such that sequences having at least a desiredlevel of complementarity will stably hybridize, while those having lowercomplementarity will not. For examples of hybridization conditions andparameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in MolecularBiology. John Wiley & Sons, Secaucus, N.J.

By “isolated”, when referring to a nucleic acid (e.g., anoligonucleotide such as RNA, DNA, or a mixed polymer) is meant a nucleicacid that is apart from a substantial portion of the genome in which itnaturally occurs and/or is substantially separated from other cellularcomponents which naturally accompany such nucleic acid. For example, anynucleic acid that has been produced synthetically (e.g., by serial basecondensation) is considered to be isolated. Likewise, nucleic acids thatare recombinantly expressed, cloned, produced by a primer extensionreaction (e.g., PCR), or otherwise excised from a genome are alsoconsidered to be isolated.

As used herein, a “fragment” means a polynucleotide that is at leastabout 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 1000nucleotides or more in length.

As used herein, “nucleic acid” refers broadly to segments of achromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acidmay be derived or obtained from an originally isolated nucleic acidsample from any source (e.g., isolated from, purified from, amplifiedfrom, cloned from, or reverse transcribed from sample DNA or RNA).

As used herein, the term “oligonucleotide” refers to a short polymercomposed of deoxyribonucleotides, ribonucleotides or any combinationthereof. Oligonucleotides are generally between about 10 and about 100nucleotides in length. Oligonucleotides are preferably 15 to 70nucleotides long, with 20 to 26 nucleotides being the most common. Anoligonucleotide may be used as a primer or as a probe.

An oligonucleotide is “specific” for a nucleic acid if theoligonucleotide has at least 50% sequence identity with a portion of thenucleic acid when the oligonucleotide and the nucleic acid are aligned.An oligonucleotide that is specific for a nucleic acid is one that,under the appropriate hybridization or washing conditions, is capable ofhybridizing to the target of interest and not substantially hybridizingto nucleic acids which are not of interest. Higher levels of sequenceidentity are preferred and include at least 75%, at least 80%, at least85%, at least 90%, or at least 95% sequence identity.

As used herein, a “primer” for amplification is an oligonucleotide thatspecifically anneals to a target or marker nucleotide sequence. The 3′nucleotide of the primer should be identical to the target or markersequence at a corresponding nucleotide position for optimal primerextension by a polymerase. As used herein, a “forward primer” is aprimer that anneals to the anti-sense strand of double stranded DNA(dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.

As used herein, the term “sample” or “test sample” refers to any liquidor solid material containing nucleic acids. In suitable embodiments, atest sample is obtained from a biological source (i.e., a “biologicalsample”), such as cells in culture or a tissue sample from an animal,most preferably, a human. In an exemplary embodiment, the sample is avaginal swab.

“Target nucleic acid” as used herein refers to segments of a chromosome,a complete gene with or without intergenic sequence, segments orportions a gene with our without intergenic sequence, or sequence ofnucleic acids to which probes or primers are designed. Target nucleicacids may include wild type sequences, nucleic acid sequences containingmutations, deletions or duplications, tandem repeat regions, a gene ofinterest, a region of a gene of interest or any upstream or downstreamregion thereof. Target nucleic acids may represent alternative sequencesor alleles of a particular gene. Target nucleic acids may be derivedfrom genomic DNA, cDNA, or RNA. As used herein target nucleic acid maybe native DNA or a PCR amplified product. In one embodiment, the targetnucleic acid is a fragment of a 16S ribosomal RNA gene from a bacterialspecies.

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds, under which nucleic acid hybridizations are conducted. Withhigh stringency conditions, nucleic acid base pairing will occur onlybetween nucleic acids that have sufficiently long segments with a highfrequency of complementary base sequences. Exemplary hybridizationconditions are as follows. High stringency generally refers toconditions that permit hybridization of only those nucleic acidsequences that form stable hybrids in 0.018M NaCl at 65° C. Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5×Denhardt's solution, 5×SSC (saline sodium citrate) 0.2%SDS (sodium dodecyl sulphate) at 42° C., followed by washing in 0.1×SSC,and 0.1% SDS at 65° C. Moderate stringency refers to conditionsequivalent to hybridization in 50% formamide, 5×Denhardt's solution,5×SSC, 0.2% SDS at 42° C., followed by washing in 0.2×SSC, 0.2% SDS, at65° C. Low stringency refers to conditions equivalent to hybridizationin 10% formamide, 5×Denhardt's solution, 6×SSC, 0.2% SDS, followed bywashing in 1×SSC, 0.2% SDS, at 50° C.

As used herein, the term “patient” refers to a subject who receivesmedical care, attention or treatment. As used herein, the term is meantto encompass a person having or suspected of having a disease includinga person who may be symptomatic for a disease but who has not yet beendiagnosed.

As used herein, the term “pathogens”, and grammatical equivalents,refers to microorganisms which are associated with disease states, e.g.,bacterial vaginosis. A pathogen may include organisms that areconsidered commensals but under certain conditions can participate in apathogenic process. Thus, pathogenic organisms that participate information of bacterial vaginosis include Gardnerella vaginalis whichunder other circumstances may be classified as a commensal. Pathogensmay be characterized by their extracellular components, e.g., proteins,etc., which are secreted, produced, or otherwise discharged by thepathogen, thereby causing the subject to be afflicted with a diseasestate associated with the pathogen. As disclosed herein, pathogensassociated with bacterial vaginosis include, but are not limited to,Gardnerella vaginalis, Atopobium vaginae, and Megasphaera spp. The term“pathogen” is also intended to encompass presently unknown infectiousagents that may be discovered in the future, since theircharacterization as a pathogen will be readily determinable by personsskilled in the art.

Assays for the Detection of Bacterial Vaginosis

Bacterial vaginosis (BV) is the most common vaginal infection in women,and is characterized by an imbalance of the normal vaginal flora. In oneaspect, the present invention provides methods for detecting thepresence or absence of bacteria associated with BV, and determining adiagnostic score based on the presence or absence of the bacteria. Whilenot wishing to be limited by theory, the presence of variousLactobacillus species is believed to be protective for BV, while thepresence of one or more pathogens, such as Gardnerella, Mobiluncus,Bacteroides, Atopobium and Megasphaera species, are believed to be someof the indicators of disease. No single one of these is necessary andsufficient to give a diagnosis of BV, however, a score based on thepresence or absence of these bacteria is useful in the diagnosis of BV(see next section).

In one embodiment, an assay for BV involves detecting nucleic acidsegments corresponding to various bacterial species that are relevant toa diagnosis of BV. Nucleic acid segments may be detected in a variety ofways, which are described in further detail below. In one embodiment, anassay for BV may be performed using PCR. In a particular embodiment, theassay for BV may be performed using a multiplex PCR format. In oneembodiment, a test is performed in two wells, both in a multiplexformat. For example, one well may include tests for lactobacilli, whilethe other contains tests for Atopobium vaginae and the genusMegasphaera. In another example, one well includes tests forlactobacilli, while the other contains tests for the pathogens Atopobiumvaginae, the genus Megasphaera, and Gardnerella vaginalis.

In one aspect, the methods described herein are designed to detectvarious lactobacilli and pathogenic species associated with BV. Assaysmay be combined in various configurations in a multiplex format. Onesubassay, referred to herein as the “Assay A”, includes one test for thedetection for the closely related species Lactobacillus acidophilus andLactobacillus crispatus, and another for the detection of Lactobacillusjensenii. All three of these species are peroxide producers andnegatively correlate to disease. Exemplary TaqMan® primers and probesfor the Peroxides assay are shown in Table 1.

TABLE 1 Exemplary Primers and Probes for Assay A Reagent BacterialSEQ ID Name Target(s) Sequence (5′ to 3′) NO: Lacto a/c L. acidophilus5′-TGCCCCATAGTCTGGGATAC-3′ 1 Forward L. crispatus Lacto a/cL. acidophilus 5′-ATGTGGCCGATCAGTCTCTC-3′ 2 Reverse L. crispatusLacto a/c L. acidophilus 5′-[Q670]-CCGGATAAGAAAGCAGATCGCATGA-[BHQ2]-3′ 3Probe L. crispatus Lacto je L. jensenii 5′-AGTAACGCGTGGGTAACCTG-3′ 4Forward Lacto je L. jensenii 5′-GTCCATCCTTTAGCGACAGC-3′ 5 ReverseLacto je L. jensenii 5′-[FAM]-CCGGATAAAAGCTACTTTCGCATGA-[BHQ1]-3′ 6Probe

A second subassay, referred to herein as the “Assay B”, includes thesame tests for L. acidophilus/crispatus and L. jensenii as the Assay A,but also includes a third test, the Lactobacillus ssp. assay, whichdetects all members of the Lactobacillus genus. Optionally, Assay B mayfurther include an assay to detect L. vaginalis. While all lactobacillihave not been shown to correlate to BV disease state, Assay B mayprovide useful information in the event that none of the peroxideproducing species from Assay A are present in a clinical sample.Exemplary primers and probes for Assay B are shown in Table 2.

TABLE 2 Exemplary Primers and Probes for Assay B Reagent BacterialSEQ ID Name Target(s) Sequence (5′ to 3′) NO: Lacto a/c L. acidophilus5′-TGCCCCATAGTCTGGGATAC-3′  1 Forward L. crispatus Lacto a/cL. acidophilus 5′-ATGTGGCCGATCAGTCTCTC-3′  2 Reverse L. crispatusLacto a/c L. acidophilus 5′-[FAM]-CCGGATAAGAAAGCAGATCGCATGA-[BHQ1]-3′  7Probe L. crispatus Lacto je L. jensenii 5′-AGTAACGCGTGGGTAACCTG-3′  4Forward Lacto je L. jensenii 5′-GTCCATCCTTTAGCGACAGC-3′  5 ReverseLacto je L. jensenii 5′-[FAM]-CCGGATAAAAGCTACTTTCGCATGA-[BHQ1]-3′  6Probe Lacto ssp Lactobacillus 5′-ACACGGCCCAAACTCCTAC-3′  8 Forward spp.Lacto ssp Lactobacillus 5′-CGATCCGAAAACCTTCTTCA-3′  9 Reverse spp.Lacto ssp Lactobacillus 5′-[Q670]-CCGAATGATGCAATCAACTTCGAG-[BHQ2]-3′ 10Probe spp. Lv L. vaginalis 5-GAGTAACACGTGGGCAACCT-3′ 20 Forward LvL. vaginalis 5′-GCCCATCCTGAAGTGATAGC-3′ 21 Reverse Lv Probe L. vaginalis5′-[FAM]-CTGAAGCGGGGGATAACATCTGGAA-3′ 22

A third subassay, referred to herein as the “Assay C,” will detectAtopobium vaginae and the genus Megasphaera. While not wishing to belimited by theory, these pathogens may correlate more highly to BVdisease than Gardnerella vaginalis and Mobiluncus sp., two agents usedto determine disease state using the Nugent score. In some embodiments,Gardnerella may be detected separately as “Assay D”. Exemplary primersand probes for the detection of pathogenic agents are shown in Table 3.

TABLE 3 Exemplary Primers and Probes for Assays C and D ReagentBacterial SEQ ID Name Target(s) Sequence (5′ to 3′) NO: Av Atopobium5′-TAGGGGAGCGAACAGGATTA-3′ 11 Forward vaginae Av Atopobium5′-CCCGTCAATTCCTTTGAGTT-3′ 12 Reverse vaginae Av Atopobium5′-[FAM]-TGGGGAGATTATACTTTCCGTGCCG-[BHQ1]-3′ 13 Probe vaginae MegaMegasphaera 5′-CACATTGGGACTGAGACACG-3′ 14 Forward spp. Mega Megasphaera5′-ACGCTTGCCACCTACGTATT-3′ 15 Reverse spp. Mega Megasphaera5′-[Q670]-ACGGTACCGTAAGAGAAAGCCACGG-[BHQ1]-3′ 16 Probe spp. GvGardnerella 5′-CTCTTGGAAACGGGTGGTAA-3′ 17 Forward spp. Gv Gardnerella5′-GAGTCTGGGCCGTATCTCAG-3′ 18 Reverse spp. Gv Probe Gardnerella5′-[Q670]-AGCTTGTAGGCGGGGTAATGGCC-[BHQ1]-3′ 19 spp.

With regard to the exemplary primers and probes, those skilled in theart will readily recognize that nucleic acid molecules may bedouble-stranded molecules and that reference to a particular site on onestrand refers, as well, to the corresponding site on a complementarystrand. In defining a variant position, allele, or nucleotide sequence,reference to an adenine, a thymine (uridine), a cytosine, or a guanineat a particular site on one strand of a nucleic acid molecule alsodefines the thymine (uridine), adenine, guanine, or cytosine(respectively) at the corresponding site on a complementary strand ofthe nucleic acid molecule. Thus, reference may be made to either strandin order to refer to a particular variant position, allele, ornucleotide sequence. Probes and primers, may be designed to hybridize toeither strand and detection methods disclosed herein may generallytarget either strand.

Determination of a Diagnostic Score

In one aspect, the present invention provides methods for diagnosingbacterial vaginosis in a subject by mathematically determining a singlediagnostic score using the levels of one or more lactobacilli and two ormore pathogenic organisms in a sample from the subject; and comparingthe diagnostic score for the individual to one or more reference scoresto determine the presence of bacterial vaginosis. In some embodiments,the single diagnostic score is determined by finding the ratio of alogarithmic function of the levels of one or more lactobacilli and alogarithmic function of the levels of two or more pathogenic organisms.

Thus, in embodiments of this aspect, an algorithm may be used todetermine a single diagnostic score. In one embodiment, an algorithm isused to determine a single diagnostic score based on cell countsmeasured in a real-time PCR assay, e.g., TaqMan®, for one or morelactobacilli and two or more pathogenic organisms. Results forlactobacilli are then subjected to a logarithmic function and divided byresults for pathogenic organisms subjected to a logarithmic function toproduce a ratio. Illustrative algorithms are presented as Algorithms 1-8below.

In some embodiments, the logarithmic functions include summing thelogarithms of the quantities of each target organism (i.e., the Sum ofLogs method). Algorithm 1, shown below, demonstrates a generic form ofthe Sum of Logs method. Illustrative Algorithms 2-5, also shown below,demonstrate exemplary embodiments of the Sum of Logs method.

In other embodiments, the logarithmic functions include taking thelogarithm of the sum of the quantities of each target organism (i.e.,the Log of Sums method). Algorithm 6, shown below, demonstrates ageneric form of the Log of Sums method. Illustrative Algorithms 7-10,shown below, demonstrate exemplary embodiments of the Logs of Sumsmethod.

While not wishing to be limited by theory, using the Sum of Logs methodemphasizes the total contribution of all organisms in the calculation,while the Log of Sums method emphasizes the most common organism.

$\begin{matrix}\frac{\sum\;{{Log}( {\mspace{11mu}\;}{{one}\mspace{14mu}{or}\mspace{14mu}{more}\mspace{14mu}{natural}\mspace{14mu}{flora}\mspace{14mu}{including}\mspace{14mu} a\mspace{14mu}{lactobacilli}} )}}{\sum\;{{Log}( {{two}\mspace{14mu}{or}\mspace{14mu}{more}\mspace{14mu}{pathogenic}{\mspace{11mu}\;}{organisms}} )}} & {{Algorithm}\mspace{14mu} 1} \\\frac{{{Log}( {{Lactobacillus}\mspace{14mu}{{spp}.}} )} + {{Log}( {{Assay}\mspace{14mu} A} )}}{\begin{matrix}{{{Log}( {{Atopobium}\mspace{14mu}{vaginae}} )} +} \\{{Log}( {{Megaphaera}\mspace{14mu}{{ssp}.}} )}\end{matrix}} & {{Algorithm}\mspace{14mu} 2} \\\frac{{{Log}( {{L.{acidophilus}}/{crispatus}} )} + {{Log}( {L\mspace{20mu}{jensenii}} )}}{\begin{matrix}{{{Log}( {{Atopobium}\mspace{14mu}{vaginae}} )} +} \\{{Log}( {{Megaphaera}\mspace{14mu}{{ssp}.}} )}\end{matrix}} & {{Algorithm}\mspace{14mu} 3} \\\frac{{{Log}( {{L.{acidophilus}}/{crispatus}} )} + {{Log}( {L\mspace{20mu}{jensenii}} )}}{\begin{matrix}{{{Log}( {{Atopobium}\mspace{14mu}{vaginae}} )} +} \\{{{Log}( {{Megaphaera}\mspace{14mu}{{ssp}.}} )} +} \\{{Log}({Gardnerella})}\end{matrix}} & {{Algorithm}\mspace{14mu} 4} \\\frac{\begin{matrix}{{{Log}( {{L.{acidophilus}}/{crispatus}} )} +} \\{{{Log}( {L\mspace{20mu}{jensenii}} )} + {{Log}( {L.{Vaginalis}} )}}\end{matrix}}{\begin{matrix}{{{Log}( {{Atopobium}\mspace{14mu}{vaginae}} )} +} \\{{{Log}( {{Megaphaera}\mspace{14mu}{{ssp}.}} )} +} \\{{Log}({Gardnerella})}\end{matrix}} & {{Algorithm}\mspace{14mu} 5} \\\frac{{Log}( {\sum\;( {{one}\mspace{14mu}{or}\mspace{14mu}{more}\mspace{14mu}{natural}\mspace{14mu}{flora}\mspace{14mu}{including}\mspace{14mu} a\mspace{14mu}{lactobacilli}}\; )} )}{{Log}( {\sum\;( {{two}\mspace{14mu}{or}\mspace{14mu}{more}\mspace{14mu}{pathogenic}{\mspace{11mu}\;}{organisms}} )} )} & {{Algorithm}\mspace{14mu} 6} \\\frac{{Log}( {{Lactobacillus}\mspace{14mu}{{spp}.{+ {Assay}}}\mspace{14mu} A} )}{{Log}( {{{Atopobium}\mspace{14mu}{vaginae}} + {{Megaphaera}\mspace{14mu}{{ssp}.}}} )} & {{Algorithm}\mspace{14mu} 7} \\\frac{{Log}( {{{L.{acidophilus}}/{crispatus}} + {L\mspace{20mu}{jensenii}}} )}{{Log}( {{{Atopobium}\mspace{14mu}{vaginae}} + {{Megaphaera}\mspace{14mu}{{ssp}.}}} )} & {{Algorithm}\mspace{14mu} 8} \\\frac{{Log}( {{{L.{acidophilus}}/{crispatus}} + {L\mspace{20mu}{jensenii}}} )}{{Log}( {{{Atopobium}\mspace{14mu}{vaginae}} + {{Megaphaera}\mspace{14mu}{{ssp}.{+ {Gardnerella}}}}} )} & {{Algorithm}\mspace{14mu} 9} \\\frac{{Log}( {{{L.{acidophilus}}/{crispatus}} + {L\mspace{20mu}{jensenii}} + {L.{vaginalis}}} )}{{Log}( {{{Atopobium}\mspace{14mu}{vaginae}} + {{Megaphaera}\mspace{14mu}{{ssp}.{+ {Gardnerella}}}}} )} & {{Algorithm}\mspace{14mu} 10}\end{matrix}$

In an exemplary embodiment, ratio values above an upper reference scoreof about 5, such as about 4.5, 4.75, 5, 5.25, and 5.5, are given adiagnosis of “Normal”, values between a lower reference score and theupper reference score are given a diagnosis of “Intermediate”. The lowerreference score is typically about 0.2, such as 0.15, 0.18, 0.2, 0.22,and 0.25. Values below the lower reference score are given a diagnosisas positive for bacterial vaginosis (“BV”).

A device may be configured to calculate a single diagnostic score andpredict the presence of bacterial vaginosis in an individual. The devicemay comprise an input interface configured to receive data, which inputinterface is in data communication with a processor, which is in datacommunication with an output interface. In various embodiments thedevice could be a handheld device, computer, a laptop, portable device,a server, or the like.

The input interface is used for entry of data including levels oflactobacilli and pathogenic organisms as determined from a sample fromthe individual. Data may be entered manually by an operator of thesystem using an input interface such as a keyboard or keypad.Alternatively, data may be entered electronically, when the inputinterface is a cable in data communication with a computer, a network, aserver, or analytical instrument. The input interface may wirelesslycommunicate with the processor.

The device further comprises a processor and a computer-readable storagemedium including computer-readable instructions stored therein that,upon execution by the processor, cause the device to compute a singlediagnostic score. In embodiments utilizing such a device, the diagnosticscore is computed using an algorithm. In some embodiments, the algorithmused to compute the single diagnostic score may comprise one or more ofillustrative Algorithms 1-10 above. In embodiments utilizing a pluralityof algorithms for determining the single diagnostic score, the resultsof the determination of each algorithm may be combined by any methodknown in the art.

In another embodiment, the device may further comprise readableinstructions (e.g. software) stored on a computer-readable storagemedium (e.g. memory) that, upon execution by the processor, compares thediagnostic score to one or more reference scores to predict the presenceof bacterial vaginosis. A diagnostic score less than a lower referencescore is predictive of bacterial vaginosis. A diagnostic score greaterthan an upper reference score value is predictive of the absence ofbacterial vaginosis. Exemplary values for use as reference scores inthese embodiments are described above. The computer-readableinstructions may be executable instructions such as program code.

In one embodiment, the data output interface, in data communication withthe processor, receives the diagnosis or the diagnostic score from theprocessor and provides the prediction or the diagnostic score to thedevice operator. The output interface may be, for example, a videodisplay monitor or a printer. The output interface may be wirelesslyconnected to the processor. In a particular embodiment, a single devicemay function as the input interface and the output interface. Oneexample of this type of interface is where the display monitor alsofunctions as a keypad or touchscreen.

In another embodiment, a semi-quantitative algorithm is used to diagnoseBV. For example, this semi-quantitative algorithm does not use acalculation, but rather considers the presence or absence of keyorganisms (see Table 13). A sample is considered normal (not indicativeof BV) if:

(1) L. acidophilus, L. crispatus, or L. jensenii are present, Atopobiumand Megasphaera are absent, and Gardnerella is present in amounts lessthan 10⁶ cells/ml; or

(2) all organisms are absent.

A sample is intermediate if:

(1) the sample contains both lactobacilli and at least one pathogen(≧10⁶ cells/ml for Gardnerella); or

(2) all organisms are absent except for Gardnerella, which is present,but is less than 10⁶ cells/ml.

A sample is considered to indicate BV if no lactobacilli are present,and at least one pathogen is present (≧10⁶ cells/ml for Gardnerella).

Sample Collection and Preparation

The methods and compositions of this invention may be used to detectnucleic acids associated with various bacteria using a biological sampleobtained from an individual. The nucleic acid (DNA or RNA) may beisolated from the sample according to any methods well known to those ofskill in the art. Biological samples may be obtained by standardprocedures and may be used immediately or stored, under conditionsappropriate for the type of biological sample, for later use.

Starting material for the detection assays is typically a clinicalsample, which is suspected to contain a lactobacillus and/or apathogenic organism. An example of a clinical sample is a vaginal swab.Next, the nucleic acids may be separated from proteins and sugarspresent in the original sample. Any purification methods known in theart may be used in the context of the present invention. Nucleic acidsequences in the sample can successfully be amplified using in vitroamplification, such as PCR. Typically, any compounds that may inhibitpolymerases are removed from the nucleic acids.

Methods of obtaining test samples are well known to those of skill inthe art and include, but are not limited to, aspirations, tissuesections, swabs, drawing of blood or other fluids, surgical or needlebiopsies, and the like. The test sample may be obtained from anindividual or patient. The test sample may contain cells, tissues orfluid obtained from a patient suspected being afflicted with bacterialvaginosis. The test sample may be a cell-containing liquid or a tissue.Samples may include, but are not limited to, cells from a vaginal swab,amniotic fluid, biopsies, blood, blood cells, bone marrow, fine needlebiopsy samples, peritoneal fluid, amniotic fluid, plasma, pleural fluid,saliva, semen, serum, tissue or tissue homogenates, frozen or paraffinsections of tissue. Samples may also be processed, such as sectioning oftissues, fractionation, purification, or cellular organelle separation.

If necessary, the sample may be collected or concentrated bycentrifugation and the like. The cells of the sample may be subjected tolysis, such as by treatments with enzymes, heat, surfactants,ultrasonication, or a combination thereof. The lysis treatment isperformed in order to obtain a sufficient amount of nucleic acid derivedfrom the bacterial cells in the same to detect using polymerase chainreaction.

Nucleic Acid Extraction and Amplification

The nucleic acid to be amplified may be from a biological sample such asa bacterial organism, cell culture, tissue sample, and the like. Variousmethods of extraction are suitable for isolating the DNA or RNA.Suitable methods include phenol and chloroform extraction. See Maniatiset al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring HarborLaboratory Press, page 16.54 (1989). Numerous commercial kits also yieldsuitable DNA and RNA including, but not limited to, QIAamp™ mini bloodkit, Agencourt Genfind™, Roche Cobas® Roche MagNA Pure® orphenol:chloroform extraction using Eppendorf Phase Lock Gels®, and theNucliSens extraction kit (Biomerieux, Marcy l'Etoile, France).

Nucleic acid extracted from cells or tissues can be amplified usingnucleic acid amplification techniques well know in the art. By way ofexample, but not by way of limitation, these techniques can include thepolymerase chain reaction (PCR), reverse transcriptase polymerase chainreaction (RT-PCR), nested PCR, ligase chain reaction. See Abravaya, K.,et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signalamplification, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S 14, (1993),amplifiable RNA reporters, Q-beta replication, transcription-basedamplification, boomerang DNA amplification, strand displacementactivation, cycling probe technology, isothermal nucleic acid sequencebased amplification (NASBA). See Kievits, T. et al., J VirologicalMethods, 35:273-286, (1991), Invader® Technology, or other sequencereplication assays or signal amplification assays. These methods ofamplification each described briefly below and are well-known in theart.

Some methods employ reverse transcription of RNA to cDNA. As noted, themethod of reverse transcription and amplification may be performed bypreviously published or recommended procedures. Various reversetranscriptases may be used, including, but not limited to, MMLV RT,RNase H mutants of MMLV RT such as Superscript and Superscript II (LifeTechnologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostablereverse transcriptase from Thermus thermophilus. For example, onemethod, but not the only method, which may be used to convert RNAextracted from plasma or serum to cDNA is the protocol adapted from theSuperscript II Preamplification system (Life Technologies, GIBCO BRL,Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian,A., PCR Methods Applic., 4:S83-S91, (1994).

LCR is a method of DNA amplification similar to PCR, except that it usesfour primers instead of two and uses the enzyme ligase to ligate or jointwo segments of DNA. LCR can be performed as according to Moore et al.,J Clin Micro, 36(4):1028-1031 (1998). Briefly, an LCR reaction mixturecontains two pair of primers, dNTP, DNA ligase and DNA polymeraserepresenting about 90 μl, to which is added 100 μl of isolated nucleicacid from the target organism. Amplification is performed in a thermalcycler (e.g., LCx of Abbott Labs, Chicago, Ill.).

TAS is a system of nucleic acid amplification in which each cycle iscomprised of a cDNA synthesis step and an RNA transcription step. In thecDNA synthesis step, a sequence recognized by a DNA-dependent RNApolymerase (i.e., a polymerase-binding sequence or PBS) is inserted intothe cDNA copy downstream of the target or marker sequence to beamplified using a two-domain oligonucleotide primer. In the second step,an RNA polymerase is used to synthesize multiple copies of RNA from thecDNA template. Amplification using TAS requires only a few cyclesbecause DNA-dependent RNA transcription can result in 10-1000 copies foreach copy of cDNA template. TAS can be performed according to Kwoh etal., PNAS, 86:1173-7 (1989). Briefly, extracted RNA is combined with TASamplification buffer and bovine serum albumin, dNTPs, NTPs, and twooligonucleotide primers, one of which contains a PBS. The sample isheated to denature the RNA template and cooled to the primer annealingtemperature. Reverse transcriptase (RT) is added the sample incubated atthe appropriate temperature to allow cDNA elongation. Subsequently T7RNA polymerase is added and the sample is incubated at 37° C. forapproximately 25 minutes for the synthesis of RNA. The above steps arethen repeated. Alternatively, after the initial cDNA synthesis, both RTand RNA polymerase are added following a 1 minute 100° C. denaturationfollowed by an RNA elongation of approximately 30 minutes at 37° C. TAScan be also be performed on solid phase as according to Wylie et al., JClin Micro, 36(12):3488-3491 (1998). In this method, nucleic acidtargets are captured with magnetic beads containing specific captureprimers. The beads with captured targets are washed and pelleted beforeadding amplification reagents which contains amplification primers,dNTP, NTP, 2500 U of reverse transcriptase and 2500 U of T7 RNApolymerase. A 100 μl TMA reaction mixture is placed in a tube, 200 μloil reagent is added and amplification is accomplished by incubation at42° C. in a waterbath for one hour.

NASBA is a transcription-based amplification method which amplifies RNAfrom either an RNA or DNA target. NASBA is a method used for thecontinuous amplification of nucleic acids in a single mixture at onetemperature. For example, for RNA amplification, avian myeloblastosisvirus (AMV) reverse transcriptase, RNase H and T7 RNA polymerase areused. This method can be performed as according to Heim, et al., NucleicAcids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction mixturecontains two specific primers, dNTP, NTP, 6.4 U of AMV reversetranscriptase, 0.08 U of E. coli Rnase H, and 32 U of T7 RNA polymerase.The amplification is carried out for 120 min at 41° C. in a total volumeof 20 μl.

In a related method, self-sustained sequence-replication (3SR) reaction,isothermal amplification of target DNA or RNA sequences in vitro usingthree enzymatic activities: reverse transcriptase, DNA-dependent RNApolymerase and E. coli ribonuclease H. This method may be modified froma 3-enzyme system to a 2-enzyme system by using human immunodeficiencyvirus (HIV)-1 reverse transcriptase instead of avian myeloblastosisvirus (AMV) reverse transcriptase to allow amplification with T7 RNApolymerase but without E. coli ribonuclease H. In the 2-enzyme 3SR, theamplified RNA is obtained in a purer form compared with the 3-enzyme 3SR(Gebinoga & Oehlenschlager Eur J Biochem, 235:256-261, 1996).

SDA is an isothermal nucleic acid amplification method. A primercontaining a restriction site is annealed to the template. Amplificationprimers are then annealed to 5′ adjacent sequences (forming a nick) andamplification is started at a fixed temperature. Newly synthesized DNAstrands are nicked by a restriction enzyme and the polymeraseamplification begins again, displacing the newly synthesized strands.SDA can be performed as according to Walker, et al., PNAS, 89:392-6(1992). Briefly, an SDA reaction mixture contains four SDA primers,dGTP, dCTP, TTP, dATP, 150 U of Hinc II, and 5 U ofexonuclease-deficient of the large fragment of E. coli DNA polymerase I(exo⁻ Klenow polymerase). The sample mixture is heated 95° C. for 4minutes to denature target DNA prior to addition of the enzymes. Afteraddition of the two enzymes, amplification is carried out for 120 min.at 37° C. in a total volume of 50 μl. Then, the reaction is terminatedby heating for 2 min. at 95° C.

The Q-beta replication system uses RNA as a template. Q-beta replicasesynthesizes the single-stranded RNA genome of the coliphage Qβ. Cleavingthe RNA and ligating in a nucleic acid of interest allows thereplication of that sequence when the RNA is replicated by Q-betareplicase (Kramer & Lizardi Trends Biotechnol. 1991 9(2):53-8, 1991).

In suitable embodiments, PCR is used to amplify a target sequence ofinterest. PCR is a technique for making many copies of a specifictemplate DNA sequence. The reaction consists of multiple amplificationcycles and is initiated using a pair of primer sequences that hybridizeto the 5′ and 3′ ends of the sequence to be copied. The amplificationcycle includes an initial denaturation, and typically up to 50 cycles ofannealing, strand elongation and strand separation (denaturation). Ineach cycle of the reaction, the DNA sequence between the primers iscopied. Primers can bind to the copied DNA as well as the originaltemplate sequence, so the total number of copies increases exponentiallywith time. PCR can be performed as according to Whelan, et al., J ofClin Micro, 33(3):556-561(1995). Briefly, a PCR reaction mixtureincludes two specific primers, dNTPs, approximately 0.25 U of Taqpolymerase, and 1×PCR Buffer.

The skilled artisan is capable of designing and preparing primers thatare appropriate for amplifying a target or marker sequence. The lengthof the amplification primers depends on several factors including thenucleotide sequence identity and the temperature at which these nucleicacids are hybridized or used during in vitro nucleic acid amplification.The considerations necessary to determine a preferred length for anamplification primer of a particular sequence identity are well-known toa person of ordinary skill. For example, the length of a short nucleicacid or oligonucleotide can relate to its hybridization specificity orselectivity.

In some embodiments, the amplification may include a labeled primerprobe, thereby allowing detection of the amplification productscorresponding to that primer or probe. In particular embodiments, theamplification may include a multiplicity of labeled primers or probes;typically, such primers are distinguishably labeled, allowing thesimultaneous detection of multiple amplification products.

In one embodiment, a primer or probe is labeled with a fluorogenicreporter dye that emits a detectable signal. While a suitable reporterdye is a fluorescent dye, any reporter dye that can be attached to adetection reagent such as an oligonucleotide probe or primer is suitablefor use in the invention. Such dyes include, but are not limited to,Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin,Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine,Rhodol Green, Tamra, Rox, and Texas Red.

In yet another embodiment, the detection reagent may be further labeledwith a quencher dye such as Tamra, Dabcyl, or Black Hole Quencher®(BHQ), especially when the reagent is used as a self-quenching probesuch as a TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,538,848) or MolecularBeacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemlessor linear beacon probe (Livak et al., 1995, PCR Method Appl., 4:357-362;Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al.,1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and6,117,635).

Nucleic acids may be amplified prior to detection or may be detecteddirectly during an amplification step (i.e., “real-time” methods). Insome embodiments, the target sequence is amplified and the resultingamplicon is detected by electrophoresis. In some embodiments, the targetsequence is amplified using a labeled primer such that the resultingamplicon is detectably labeled. In some embodiments, the primer isfluorescently labeled.

In one embodiment, detection of a target nucleic acid, such as a nucleicacid from a lactobacillus or pathogenic bacteria, is performed using theTaqMan® assay, which is also known as the 5′ nuclease assay (U.S. Pat.Nos. 5,210,015 and 5,538,848). The TaqMan® assay detects theaccumulation of a specific amplified product during PCR. The TaqMan®assay utilizes an oligonucleotide probe labeled with a fluorescentreporter dye and a quencher dye. The reporter dye is excited byirradiation at an appropriate wavelength, it transfers energy to thequencher dye in the same probe via a process called fluorescenceresonance energy transfer (FRET). When attached to the probe, theexcited reporter dye does not emit a signal. The proximity of thequencher dye to the reporter dye in the intact probe maintains a reducedfluorescence for the reporter. The reporter dye and quencher dye may beat the 5′ most and the 3′ most ends, respectively or vice versa.Alternatively, the reporter dye may be at the 5′ or 3′ most end whilethe quencher dye is attached to an internal nucleotide, or vice versa.In yet another embodiment, both the reporter and the quencher may beattached to internal nucleotides at a distance from each other such thatfluorescence of the reporter is reduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget-containing template which is amplified during PCR.

TaqMan® primer and probe sequences can readily be determined using thevariant and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the target nucleic acids areuseful in diagnostic assays for BV and related pathologies, and can bereadily incorporated into a kit format. The present invention alsoincludes modifications of the TaqMan® assay well known in the art suchas the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and6,117,635). Exemplary TaqMan® primers and probes for various targetnucleic acids are shown in Tables 1, 2, and 3.

In an illustrative embodiment, real time PCR is performed using TaqMan®probes in combination with a suitable amplification/analyzer such as theABI Prism® 7900HT Sequence Detection System. The ABI PRISM® 7500Sequence Detection System is a real-time PCR system that detects andquantitates nucleic acid sequences. Real time detection on the ABI Prism7500 or 7500 Sequence Detector monitors fluorescence and calculates Rnduring each PCR cycle. The threshold cycle, or Ct value, is the cycle atwhich fluorescence intersects the threshold value. The threshold valueis determined by the sequence detection system software or manually. TheCt can be correlated to the initial amount of nucleic acids or number ofstarting cells using a standard curve.

Other methods of probe hybridization detected in real time can be usedfor detecting amplification a target or marker sequence flanking atandem repeat region. For example, the commercially available MGBEclipse™ probes (Epoch Biosciences), which do not rely on a probedegradation can be used. MGB Eclipse™ probes work by ahybridization-triggered fluorescence mechanism. MGB Eclipse™ probes havethe Eclipse™ Dark Quencher and the MGB positioned at the 5′-end of theprobe. The fluorophore is located on the 3′-end of the probe. When theprobe is in solution and not hybridized, the three dimensionalconformation brings the quencher into close proximity of thefluorophore, and the fluorescence is quenched. However, when the probeanneals to a target or marker sequence, the probe is unfolded, thequencher is moved from the fluorophore, and the resultant fluorescencecan be detected.

Oligonucleotide probes can be designed which are between about 10 andabout 100 nucleotides in length and hybridize to the amplified region.Oligonucleotides probes are preferably 12 to 70 nucleotides; morepreferably 15-60 nucleotides in length; and most preferably 15-25nucleotides in length. The probe may be labeled. Amplified fragments maybe detected using standard gel electrophoresis methods. For example, insome embodiments, amplified fractions are separated on an agarose geland stained with ethidium bromide by methods known in the art to detectamplified fragments.

Internal Control Nucleic Acids

As a quality control measure, an internal amplification control may beincluded in one or more samples to be extracted and amplified. Theskilled artisan will understand that any detectable sequence that is notderived from the target bacterial species can be used as the controlsequence. A control sequence can be produced synthetically. If PCRamplification is successful, the internal amplification controlamplicons can then be detected. Additionally, if included in the sampleprior to purification of nucleic acids, the control sequences can alsoact as a positive purification control.

Kits

In a further aspect, the invention disclosure provides kits fordiagnosing BV in an individual, the kit comprising: a set of reagentsfor determining the presence or absence, or differential presence, ofone or more bacteria indicative of BV. In one embodiment, the kitcontains a set of nucleic acid primers for detecting one or morelactobacilli and two or more pathogenic organisms in a sample. Forexample, the kit may comprise a primer pair for amplifying a fragment ofa nucleic acid from one or more lactobacilli and primer pairs foramplifying fragments of nucleic acids two or more pathogenic organisms.In one embodiment, at least one primer pair is selected from the groupconsisting of: SEQ ID NOs: 1/2, SEQ ID NOs: 4/5, SEQ ID NOs: 8/9; SEQ IDNOs: 11/12; SEQ ID NOs: 14-15; and SEQ ID NOs: 17/18. In exemplaryembodiments, the kit contains one or more of the primers or probes ofSEQ ID NOs: 1-19.

EXAMPLE

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way.

In this Example, single and multiplex PCR assays for various bacteriaassociated with bacterial vaginosis were conducted. The “subassays” inthis study used specific target primer/probe sets for amplification anddetection of DNA extracts using TaqMan® technology. The primershybridized to flanking regions within the 16S ribosomal RNA gene of thetarget species, but did not bind to the same region. The probes used fordetection of the amplicon are labeled with a 5′ reporter dye and a 3′quencher dye and binds to a sequence of the 16S gene, which is unique tothe appropriate species.

Materials and Methods

Vaginal samples from patients were collected using the Aptima swabtransport media. Swab transport media is included in the Aptima VaginalSwab Collection Kit (Gen-Probe, Catalog No. 1162). The samples weresubjected to a sample preparation in which the nucleic acids werereleased and purified from the other components of the sample usingMagNAPure™ LC DNA isolation kit III (Roche Diagnostics GmbH, Germany,Cat No. 3 264 785). The sample preparation yielded a specimen containingthe nucleic acids in elution buffer.

The primer and probe set for Lactobacillus acidophilus/crispatus weredesigned to detect the closely related acidophilus and crispatus speciesof the Lactobacillus genus. The sets for Atopobium vaginae, L.,jensenii, and L. vaginalis were specific to the given species, and didnot detect other members of genus. The sets for Lactobacillus ssp,Megasphaera, Gardnerella and Mobiluncus were designed to detect allmembers of the genus. In Assay A, the probes used to detect both the L.acidophilus/crispatus and L. jensenii species were labeled with the FAMreporter dye. This means that while all three species can be detected byan increase in FAM fluorescence, concentrations of the three speciescannot be distinguished. Thus, the peroxide producers detected by themultiplex assay may be referred to in this study as Lactobacillusacidophilus/crispatus/jensenii. The primers and probes for each speciesare shown in Tables 1-3 above.

To ensure the absence of PCR inhibitors in a sample, an internalpositive amplification control (IPC) is included with each specimen. Thepositive control primers and probe are added to create a multiplexreaction with the target and sample primers. The IPC amplicon isdetected with a probe labeled with VIC or JOE as the 5′ reporter dye. Asample can be interpreted as negative only if the analysis of theinternal positive control indicates that DNA amplification has occurredin the reaction tube. The reaction mixtures for the Assays A, C, and Dare shown in Tables 4-6 below, respectively.

TABLE 4 Assay A PCR Mix Unit of Final uL per Measure Concentrationreaction 1000 rxns. Per reaction Sterile Nuclease Free Water 8.39 8.39mL Lacto a/c-F (100 uM) 0.25 250 uL 500 nM Lacto a/c-R (100 uM) 0.25 250uL 500 nM Lacto a/c-P Q670/BHQ2 0.05 50 uL 100 nM (100 uM) Lacto je-F(100 uM) 0.25 250 uL 500 nM Lacto je-R (100 uM) 0.25 250 uL 500 nM Lactoje-P (100 uM) 0.05 50 uL 100 nM 10x QIPC2 Mix 5 5.0 mL 1x   (VIC/NFQ)50x QIPC2 DNA 0.01 10 uL 0.01x Total 14.5 14.5 mL

TABLE 5 Assay C PCR Mix Unit of Final uL per Measure Concentrationreaction 1000 rxns. Per reaction Sterile Nuclease Free Water 8.39 8.39mL Av-F (100 uM) 0.25 250 uL 500 nM Av-R (100 uM) 0.25 250 uL 500 nMAv-P (100 uM) 0.05 50 uL 100 nM Mega-F (100 uM) 0.25 250 uL 500 nMMega-R (100 uM) 0.25 250 uL 500 nM Mega-P (100 uM) 0.05 50 uL 100 nM 10xQIPC2 Mix 5 5.0 mL 1x   (VIC/NFQ) 50x QIPC2 DNA 0.01 10 uL 0.01x Total14.5 14.5 mL

TABLE 6 Assay D PCR Mix Unit of Final uL per Measure Concentrationreaction 1000 rxns. Per reaction Sterile Nuclease Free Water 8.94 8.94mL Gv-F (100 uM) 0.25 250 uL 500 nM Gv-R (100 uM) 0.25 250 uL 500 nMGv-P (100 uM) 0.05 50 uL 100 nM 10x QIPC2 Mix 5 5.0 mL 1x   (JOE/EDQ)50x QIPC2 DNA 0.01 10 uL 0.01x Total 14.5 14.5 mL

Master mixes were assembled by taking 350 μL of each PCR mix prepared asshown in Tables 4-6 above and combining with 604 μL of TaqMan® Universal2×PCR Master mix and 12 μL AmpliTaq Gold® DNA polymerase. Cyclingparameters for the assay were: 50° C. for 2 min, 95° C. for 10 min, 50cycles of 95.0° C. for 15 sec to 60° C. for 1 min.

The Amplitaq Gold® polymerase used to amplify the target DNA includes a5′ to 3′ exonuclease activity which degrades the bound probe andphysically separates the reporter from the quencher dyes, resulting inan increased fluorescent signal. Increased fluorescence is plottedagainst the PCR cycle. The PCR cycle at which the plot line crosses achosen cutoff is called the Cycle Threshold (Ct). This is the standardunit of measure in TaqMan® based real-time PCR assays. A lower Ct valueindicates an earlier exponential phase for a reaction, and is correlatedto a higher initial concentration. The Ct values were compared to astandard curve to give quantitative data of cell concentrations in theoriginal sample.

Results

The Bacterial Vaginosis PCR Assay described above was compared to thetraditional Nugent Score procedure for the diagnosis of BV. Sixty-nine(69) patient samples were analyzed both by a microscopic determinationof Nugent Score, and by the Bacterial Vaginosis PCR Assay. Quantitativeresults determined from the Bacterial Vaginosis PCR Assay for patientsamples is shown in Table 7.

TABLE 7 Organism Detection by Assay (Units are Log (Cells/ml)) Assay AAssay B Individual Assays Lacto Lacto Lacto Lacto Assay C Mobil. NugentSample a/c je. ssp. a/c/je A. vaginae Megasphaera L. vagin. Gardnerellassp. Score 1 5.4 3.7 6.3 6.4 4 2 7.1 6.9 7.0 7.0 1.2 0 3 6.8 6.4 2.4 0 46.6 6.5 6.5 0 5 6.9 6.4 6.3 2.8 0 6 4.7 3.6 1 7 5.1 3.7 5 4.9 1 8 3.06.8 6.1 6.1 7.3 3.6 0 9 3.7 8 10 6.3 5.9 6.3 6.3 8.0 3.3 1.2 0 11 5.77.3 6.4 6 12 5.7 5.7 5.2 3.7 0 13 7.8 7.0 7.1 6.4 6 14 5.5 6.9 6.5 6.58.5 0 15 6.1 5 16 4.2 7.7 8.1 7.8 5 17 7.1 6.3 6.5 3 18 5.4 2.7 0 19 6.27.2 8.5 7.9 4.3 8 20 5.9 6.2 0 21 6.5 6.1 6.5 6.5 6.3 4.1 3.0 0 22 4 233.9 2.6 1 24 4.1 6.2 4.6 2.0 8 25 6.4 0 26 5.7 2.8 0 27 2.0 6.8 5 28 029 6.4 6.1 6.3 6.3 1 30 4.4 6.8 7.7 7.3 6.7 8 31 7.1 6.6 6.9 7.0 8.3 1.10 32 4.4 4.2 1 33 2.7 4 34 6.2 3.3 8.4 7.7 7.4 4 35 6.0 5.8 5.8 4.9 2.71 36 4.7 3.8 1 37 7.1 7.3 7.0 7.0 5.1 0 38 6.5 7.0 6.6 6.6 0 39 6.0 5.63.9 0 40 5.1 1.7 0 41 5.1 4.3 3.7 0 42 5.9 5.3 4.4 1.6 3 43 4.5 2.5 6 446.5 6.6 6.5 6.5 7.7 3.7 1 45 6.3 7.9 7 7.1 7.4 1.8 5 46 6.1 6.7 6.2 6.21 47 4.9 6 48 5.7 2 49 5.9 5.8 5.7 5.7 1.3 0 50 7.3 6.5 6.6 5.8 1.3 0 515.5 6.1 7.8 7.6 8 52 5.0 4 53 5.5 0 54 2.3 4.8 6.3 6 55 2.2 6 56 6.6 6.16.0 0 57 5.1 4.3 6.2 5.5 1 58 6.4 6.2 6.3 2.4 0 59 5.8 6.3 5.9 6.0 0 604.7 2.7 1 61 4.9 4.9 4.7 4.2 8 62 3.4 5.1 0.5 4.5 8.2 6.4 3.6 0 63 6.76.0 6.0 0 64 5.3 0 65 6.3 6.2 6.2 7.0 0 66 4.7 2.2 4 67 6.3 5.5 6.2 6.19.1 6.6 0 68 6.8 6.1 6.1 3.0 0 69 6.2 6.0 6.0 0

The samples presented in Table 7 were categorized based on Nugent Score.FIG. 1 shows the percent of swab specimens containing bacterial agentsas arranged by Nugent Score. For some organisms, in particularLactobacillus ssp. and Mobiluncus, the percent of specimens containingthese organisms do not differ dramatically given disease state. However,dramatic differences exist for the peroxide-producing lactobacilli(Lactobacillus acidophilus/crispatus, L. jensenii, and L. vaginalis).Dramatic increases also exist for A. vaginae, Megasphaera ssp., andGardnerella. In this study, Atopobium and Megasphaera both appear in 57%of samples given a diagnosis of BV based on Nugent score. Gardnerellaappears in 85% of BV samples.

FIG. 2 shows the mean quantities of bacterial agents as arranged byNugent Score. Samples in which none of a given agent was detected werenot included in statistics. Error bars show ±2 standard deviations. Formost organisms, a broad overlap exists between cell counts for thedifferent disease states. More dramatic differences in mean existbetween disease states for Atopobium, Megasphaera, Gardnerella, L.acidophilus/crispatus, L. jensenii, and L. vaginalis.

Analysis of BV PCR Test Results Using Algorithms

Algorithms 1-8 (described above) were used to create a ratio based onthe quantity of various organisms in a vaginal swab sample from apatient. Table 8 shows the ratios produced by each algorithm. Algorithms1-4 were derived by adding the Logs of the quantities for each targetorganism (Sum of Logs). Algorithms 5-8 were derived by adding thequantities first, and then taking the Log of the result (Log of Sums).

TABLE 8 Analysis of BV PCR Test Results Using Algorithms AlgorithmNugent Semi Sum of Logs Log of Sums Sample Score Quant 1 2 3 4 5 6 7 8 14 BV 0.53 0.10 0.06 0.06 0.85 0.16 0.15 0.15 2 0 Norm 14.05 13.91 13.9113.91 7.32 7.27 7.27 7.27 3 0 Norm 12.83 6.85 6.85 6.85 6.72 6.85 6.856.85 4 0 Norm 13.00 6.56 6.56 6.56 6.8 6.56 6.56 6.56 5 0 Norm 12.706.89 6.89 6.89 6.66 6.89 6.89 6.89 6 1 Int 4.69 1.00 1.00 1.00 4.69 1.001.00 1.00 7 1 Norm 9.85 8.8 8.8 8.8 5.23 5.08 5.08 5.08 8 0 Norm 12.219.75 9.75 17.05 6.41 6.77 6.77 7.41 9 8 Norm 1.00 1.00 1.00 1.00 1.001.00 1.00 1.00 10 0 Norm 12.6 12.2 3.68 6.08 6.6 6.44 1.94 2.41 11 6 BV5.66 1.00 0.16 1.14 5.66 1.00 0.16 1.14 12 0 Norm 10.94 5.71 5.71 9.465.85 5.71 5.71 5.71 13 6 Int 14.04 7.80 1.22 1.22 7.33 7.80 1.22 1.22 140 Norm 12.95 12.41 12.41 20.88 6.77 6.95 6.95 8.48 15 5 Norm 6.10 1.001.00 1.00 6.10 1.00 1.00 1.00 16 5 BV 0.27 0.06 0.04 0.04 0.51 0.12 0.120.12 17 3 Norm 12.07 7.12 7.12 7.12 6.70 7.12 7.12 7.12 18 0 Norm 5.421.00 1.00 1.00 5.42 1.00 1.00 1.00 19 8 BV 0.39 0.06 0.04 0.04 0.73 0.120.12 0.12 20 0 Norm 5.89 1.00 1.00 6.17 5.89 1.00 1.00 1.00 21 0 Norm13.07 12.57 3.09 4.65 6.83 6.62 1.63 1.67 22 4 Norm 1.00 1.00 1.00 1.001.00 1.00 1.00 1.00 23 1 Norm 3.93 1.00 1.00 1.00 3.93 1.00 1.00 1.00 248 Norm 6.16 4.12 0.91 0.91 0.16 4.12 0.91 0.91 25 0 Norm 6.45 1.00 1.001.00 6.45 1.00 1.00 1.00 26 0 Norm 5.69 1.00 1.00 1.00 5.69 1.00 1.001.00 27 5 BV 2.04 1.00 0.15 0.15 2.04 1.00 0.15 0.15 28 0 Norm 1.00 1.001.00 1.00 1.00 1.00 1.00 1.00 29 1 Norm 12.51 12.46 12.46 12.46 6.566.55 6.55 6.55 30 8 BV 0.31 0.07 0.05 0.05 0.57 0.13 0.13 0.13 31 0 Norm13.90 13.70 13.70 22.03 7.25 7.20 7.20 8.36 32 1 Norm 4.41 1.00 1.001.00 4.41 1.00 1.00 1.00 33 4 Norm 1.00 1.00 1.00 1.00 1.00 1.00 1.001.00 34 4 BV 0.53 0.09 0.05 0.40 0.74 0.12 0.12 0.91 35 1 Norm 11.676.02 1.24 1.24 6.14 6.02 1.24 1.24 36 1 Norm 4.71 1.00 1.00 1.00 4.711.00 1.00 1.00 37 0 Norm 14.02 14.36 14.36 19.46 7.31 7.49 7.49 7.49 380 Norm 13.23 13.56 13.56 13.56 6.92 7.16 7.16 7.16 39 0 Int 6.02 1.000.18 0.18 6.02 1.00 0.18 0.18 40 0 Norm 5.14 1.00 1.00 1.00 5.14 1.001.00 1.00 41 0 Norm 5.08 1.00 0.23 0.23 5.08 1.00 0.23 0.23 42 3 Norm5.89 1.00 0.23 1.19 5.89 1.00 0.23 1.19 43 6 Norm 4.52 1.00 1.00 1.004.52 1.00 1.00 1.00 44 1 Norm 12.97 13.15 13.15 20.84 6.79 6.88 6.887.76 45 5 Int 14.08 14.19 1.92 1.92 7.34 7.90 1.07 1.07 46 1 Norm 12.4312.77 12.77 12.77 6.52 6.78 6.78 6.78 47 6 Norm 4.87 1.00 1.00 1.00 4.871.00 1.00 1.00 48 2 Norm 5.70 1.00 1.00 1.00 5.70 1.00 1.00 1.00 49 0Norm 11.52 5.88 5.88 11.60 6.07 5.88 5.88 6.11 50 0 Norm 13.13 7.31 7.3113.10 6.87 7.31 7.31 7.32 51 8 BV 0.40 0.07 0.05 0.05 0.70 0.12 0.120.12 52 4 Int 1.00 1.00 0.20 0.20 1.00 1.00 0.20 0.20 53 0 Norm 5.481.00 1.00 1.00 5.48 1.00 1.00 1.00 54 6 BV 0.48 0.21 0.09 0.09 0.48 0.210.16 0.16 55 6 Norm 1.00 1.00 0.46 0.46 1.00 1.00 0.46 0.46 56 0 Norm12.01 6.61 6.61 6.61 6.31 6.61 6.61 6.61 57 1 BV 5.13 1.00 0.16 0.705.13 1.00 0.16 0.70 58 0 Norm 12.54 6.39 6.39 6.39 6.57 6.30 6.39 6.3959 0 Norm 11.90 12.07 12.07 12.07 6.26 6.39 6.39 6.39 60 1 Norm 4.731.70 1.70 1.70 4.73 1.70 1.70 1.70 61 8 Int 9.6 4.88 1.15 1.15 5.11 4.881.15 1.15 62 0 Int 1.24 0.75 0.31 1.06 1.13 0.75 0.53 1.27 63 0 Norm12.01 6.67 6.67 6.67 6.3 6.67 6.67 6.67 64 0 Norm 5.29 1.00 1.00 1.005.29 1.00 1.00 1.00 65 0 Norm 12.42 6.32 6.32 13.35 6.51 6.32 6.32 7.1066 4 Norm 4.74 1.00 1.00 1.00 4.74 1.00 1.00 1.00 67 0 Int 12.25 11.781.78 3.17 6.43 6.34 0.95 1.38 68 0 Norm 12.20 6.79 6.79 6.79 6.40 6.796.79 6.79 69 0 Norm 12.03 6.19 6.19 6.19 6.32 6.19 6.19 6.19

Table 9 shows the division of samples into the three disease classes byboth the Nugent Score and the score determined from the BacterialVaginosis PCR test using Algorithm 6, which includes data from Assay A(L. acidophilus/crispatus and L. jensenii) and Assay C (Atopobiumvaginae and Megasphaera ssp.). Ratio results from 0 to 0.199 were givena diagnosis of BV, 0.2 to 4.99 a diagnosis of intermediate, and 5 andabove a diagnosis of normal.

TABLE 9 Concordance between Nugent Score Results and Results fromBacterial Vaginosis Assay by PCR. Diagnosis Using PCR Assay for BV(Algorithm 6) BV Intermediate Normal Nugent BV 4 3 0 7 ScoreIntermediate 2 11 2 15 Normal 0 19 28 47 6 33 30 Sensitivity: 57%Specificity: 60% Concordance: 62%

The sensitivity refers to the detection of BV by the PCR assay ofsamples that were also positive for BV by the Nugent Score. Thespecificity refers to the determination of normal samples by PCR thatwere also normal by a Nugent Score. Total concordance (agreement for allthree classes divided by the total samples) was 62%.

Table 10 also includes data for Gardnerella in the diagnostic score. Theresults for this table were derived using Algorithm 7 which includesdata from Assay A, Assay C and Assay D. Ratio results from 0 to 0.199were given a diagnosis of BV, 0.2 to 4.99 a diagnosis of intermediate,and 5 and above a diagnosis of normal.

TABLE 10 Concordance between Nugent Score Results and Results fromBacterial Vaginosis Assay by PCR including Gardnerella data. DiagnosisUsing PCR Assay for BV (Algorithm 7) BV Intermediate Normal Nugent BV 43 0 7 Score Intermediate 5 10 0 15 Normal 1 22 24 47 10 35 24Sensitivity: 57% Specificity: 51% Concordance: 55%

Table 11 includes data for both Gardnerella and L. vaginalis. Thedetermination of a diagnostic score used Algorithm 8. Ratio results from0 to 0.199 were given a diagnosis of BV, 0.2 to 4.99 a diagnosis ofintermediate, and 5 and above a diagnosis of normal.

TABLE 11 Concordance between Nugent Score Results and Results fromBacterial Vaginosis Assay by PCR including Gardnerella and L. vaginalisdata. Diagnosis Using PCR Assay for BV (Algorithm 8) BV IntermediateNormal Nugent BV 4 3 0 7 Score Intermediate 3 12 0 15 Normal 0 22 25 477 37 25 Sensitivity: 57% Specificity: 53% Concordance: 59%

The sensitivity refers to the detection of BV by the PCR assay ofsamples that were also positive for BV by the Nugent Score. Thespecificity refers to the determination of normal samples by PCR thatwere also normal by a Nugent Score. Concordance using Gardnerella withor without L. vaginalis was 55% and 59%, respectively.

Table 12 shows concordance between Nugent Score results and results fromBacterial Vaginosis by PCR using a semi-quantitative algorithm. Thesemi-quantitative algorithm considers the presence or absence of keyorganisms. A sample is considered normal (not indicative of BV) if: (1)L. acidophilus, L. crispatus, or L. jensenii are present, Atopobium andMegasphaera are absent, and Gardnerella is present in amounts less than10⁶ cells/ml; or (2) all organisms are absent. A sample is intermediateif: (1) the sample contains both lactobacilli and at least one pathogen(>10⁶ cells/ml for Gardnerella); or (2) all organisms are absent exceptfor Gardnerella, which is present, but is less than 10⁶ cells/ml, seeTable 13. The concordance using the semi-quantitative algorithm was 71%.

TABLE 12 Concordance between Nugent Score Results and Results fromBacterial Vaginosis Assay by PCR Using a Semi-Quantitative Algorithm.Diagnosis Using PCR Assay for BV (Semi-Quantitative Algorithm) BVIntermediate Normal Nugent BV 4 1 2 7 Score Intermediate 5 3 7 15 Normal1 4 42 47 10 8 51 Sensitivity: 57% Specificity: 89% Concordance: 71%

TABLE 13 Classification of Subjects based on Detection of VariousBacteria. Normal Intermediate BV L. acidophilus/crispatus or L.jensenii + − + − − Atopobium or Megasphaera − − + − + or Gardnerella<6.0 − ≧6.0 <6.0 ≧6.0

Analysis of Method Comparison Data

Significant differences between disease states as determined by NugentScore were seen in percent positivity for all organisms except forLactobacillus ssp. and Mobiluncus ssp. (FIG. 1). For average cellcounts, differences between cell counts for samples with differentNugent Scores were most dramatic for Atopobium, Megasphaera, L.acidophilus/crispatus, L. jensenii, and L. vaginalis (FIG. 2). Thedifferences more modest for Gardnerella and Mobiluncus (FIG. 2).

Similar disease state diagnoses were obtained with both the Sum of Logsand Log of Sums methods of calculating ratios. While these measures cantheoretically produce differing disease state calls, they do not do soin this study. The semi-quantitative algorithm may also be used todetermine a diagnosis.

These results demonstrate that the BV Real-Time PCR Assay provides atleast two distinct advantages over the Nugent Score as a method ofdiagnosis. First, the assay is able to distinguish peroxide producinglactobacilli from other species, which the Nugent Score method does notdo. Second, these results support a role for Atopobium and Megasphaerain BV, which are not detected in the Nugent Score analysis. Thedetection of these agents by the current assay will aid in diagnosis andcontribute further information to the ongoing discussion of agents whichcause BV.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All nucleotide sequencesprovided herein are presented in the 5′ to 3′ direction.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed.

Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification, improvement and variation of the inventionsembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications, improvements and variations areconsidered to be within the scope of this invention. The materials,methods, and examples provided here are representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

Other embodiments are set forth within the following claims.

What is claimed is:
 1. A method for identifying bacterial vaginosis in ahuman female subject, the method comprising: (a) obtaining a vaginalswab sample from the subject; (b) measuring levels of Atopobium vaginae,Megasphaera genus and one or more Lactobacilli species selected from thegroup consisting of Lactobacillus acidophilus, Lactobacillus crispatus,and Lactobacillus jensenii in the sample, wherein the levels ofAtopobium vaginae, Megasphaera genus and of one or more Lactobacillispecies are detected with one or more oligonucleotides capable ofspecifically hybridizing to a target nucleic acid sequence fromAtopobium vaginae, Megasphaera genus, Lactobacillus acidophilus,Lactobacillus crispatus, and/or Lactobacillus jensenii, wherein levelsof Gardnerella vaginalis and Mobiluncus sp are not measured; wherein thelevels are determined with one or more oligonucleotide primers selectedfrom SEQ ID NOs: 1, 2, 4, 5, 11, 12, 14 and 15, and complements thereof;(c) calculating a diagnostic score as the ratio of a logarithmicfunction of the levels of the one or more Lactobacilli species and alogarithmic function of the levels of the Atopobium vaginae andMegasphaera genus; and (d) identifying the subject as having bacterialvaginosis with the diagnostic score lower than a reference score.
 2. Amethod for identifying bacterial vaginosis in a human female subject,the method comprising: (a) obtaining a vaginal swab sample from thesubject; (b) measuring levels of Atopobium vaginae, Megasphaera genusand one or more Lactobacilli species selected from the group consistingof Lactobacillus acidophilus, Lactobacillus crispatus, and Lactobacillusjensenii in the sample, wherein the levels of Atopobium vaginae,Megasphaera genus and of one or more Lactobacilli species are detectedwith one or more oligonucleotides capable of specifically hybridizing toa target nucleic acid sequence from Atopobium vaginae, Megasphaeragenus, Lactobacillus acidophilus, Lactobacillus crispalus, and/orLactobacillus jensenii, wherein levels of Gardnerella vaginalis andMobiluncus sp are not measured; wherein the levels are determined by PCRwith oligonucleotide primer pairs comprising SEQ ID NOs: 1, 2, 4, 5, 11,12, 14 and 15; (c) calculating a diagnostic score as the ratio of alogarithmic function of the levels of the one or more Lactobacillispecies and a logarithmic function of the levels of the Atopobiumvaginae and Megasphaera genus; and (d) identifying the subject as havingbacterial vaginosis with the diagnostic score lower than a referencescore.